PROJECT SUMMARY/ABSTRACT Transcription in all bacteria is achieved by a single core RNA polymerase (RNAP) which associates with a ?-subunit to form an RNAP holoenzyme, bind DNA promoter sequences, and initiate transcription. Most transcriptional regulation occurs at the level of initiation and transcription factors mediate this regulation by directly modulating the interaction between RNAP and the promoter, manipulating the rates of interconversion between closed and open RNAP-promoter complexes (RPc and RPo respectively), or affecting the rate of promoter escape. We have recently shown that transcription in Mycobacterium tuberculosis (Mtb) is considerably different from that in the model bacterium E. coli in that Mtb RNAP forms inherently unstable RPo complexes as compared to E. coli RNAP. Furthermore, Mtb possess two essential transcription factors, CarD and RbpA, that are absent from E. coli. We have previously shown that these factors stabilize mycobacterial RPo, albeit through different mechanisms, and are able to cooperatively and dramatically change the kinetics of Mtb RNAP RPo formation such that it mirrors those of E. coli RNAP. We have been studying CarD and RbpA activities in vivo and in vitro and have proposed kinetic mechanisms for each factor in the context of the housekeeping ?A RNAP holoenzyme on the ribosomal RNA (rRNA) rrnAP3 promoter. Our studies have been instrumental in understanding the fundamental properties of these essential transcription factors and have revealed insight into unique properties of Mtb transcription. However, CarD and RbpA activities have only been examined on a handful of mycobacterial promoters with sequence elements similar to promoters found in E. coli even though in general Mtb promoters differ considerably from those in E. coli. In addition, CarD has only been studied in the context of the ?A RNAP holoenzyme, despite the importance of the 12 Mtb alternative ?-factors that regulate the bacterias response to stresses encounter during pathogenesis. Thus, we still do not know how CarD and RbpA affect expression of the vast majority of genes within the Mtb chromosome and how this regulation contributes to viability under the conditions Mtb experiences during infection. In this project, we will measure the kinetics of Mtb initiation using rapid-mixing stopped-flow techniques as well as high-resolution single-molecule approaches to address how CarD and RbpA differentially affect gene expression from diverse promoters and in the context of alternative holoenzymes essential for bacterial stress responses enacted during infection. Our Aims are to: (1) Test the hypothesis that CarD and RbpA can either activate or repress transcription depending on promoter context, (2) Expand our kinetic model of factor-dependent mycobacterial transcription initiation, and (3) Determine how CarD and RbpA are influenced by alternative sigma-factors. Completion of these Aims will result in a holistic view of Mtb transcriptional regulation genome-wide and will expand paradigms of prokaryotic transcription beyond traditional model systems. These studies will also provide insight into mechanisms of Mtb pathogenesis that may inform the development of future therapeutic strategies.